| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Physiology and Biochemistry |
Department of Botany, University of Vermont, Burlington, Vermont 05405-0086 USA
Received for publication March 15, 2005. Accepted for publication July 11, 2005.
ABSTRACT
Legumes are unique among higher plants in forming a symbiosis with Rhizobium. Phylogenetic studies indicate this symbiosis may have evolved as many as three times within the Fabaceae; alternatively, a predisposition for nodulation evolved early in the history of the legume lineage. We have identified a physiological trait—increased lateral root formation in response to abscisic acid (ABA)— that marks all nodulating and non-nodulating legume species in our study set with the exception of Chamaecrista fasciculata and Cercis occidentalis. In contrast, nonlegume species tested decrease lateral root formation in response to ABA. Cercis is not a descendant of any common ancestor hypothesized to have evolved Rhizobium nodulation and has an intermediate response to ABA, partway between that of nonlegumes and legumes. We suggest that acquisition of altered responsiveness of roots to ABA is coincident with the appearance of a predisposition for nodulation within the legumes, followed by a loss in Chamaecrista. In addition, we demonstrate that altered ABA responsiveness of lateral root formation characterizes roots of the actinorhizal nodulator, Casuarina glauca, but not the closely related, nonactinorhizal species, Betula papyrifera. Thus our data provide evidence for a physiological root trait associated with nodulation both in legumes and in an actinorhizal plant.
Key Words: abscisic acid • actinorhizal • Arabidopsis thaliana • Fabaceae • lateral root • Medicago truncatula • nodulation • Rhizobium
Root branches, or lateral roots, are an integral component of root-system architecture and play an important role in enlarging the root system to facilitate the absorption of water and micro- and macronutrients. Lateral root growth and development is greatly influenced by complex interactions among different hormonal, developmental, and environmental factors (Casimiro et al., 2003
; López-Bucio et al., 2003
; Malamy, 2005
). Understanding those factors affecting lateral root formation is crucial for maximizing crop production. One important factor that integrates growth and development with responses to the environment is the sesquiterpenoid hormone abscisic acid (ABA) (Signora et al., 2001
; Sharp and LeNoble, 2002
).
ABA is a small, lipophilic plant hormone, originally believed to be involved solely in promoting seed maturation and response to water stress. ABA levels accumulate in the seed by mid to late embryogenesis, where it controls many processes, such as embryo morphogenesis, storage-protein synthesis, desiccation tolerance, and the onset and maintenance of dormancy (Giraudat et al., 1994
). In vegetative tissues, ABA mediates responses to abiotic stresses such as drought, salt, and cold. ABA can induce short-term responses such as stomatal closure—minimizing transpirational water loss—or long-term responses involving changes in gene expression (Himmelbach et al., 1998
; Finkelstein et al., 2002
).
Until recently, ABA was thought to play a role in root growth solely during water stress (Spollen et al., 2000
). However, several studies in Arabidopsis thaliana (L.) Heynh point to a function for ABA in normal root development. The growth retardation phenotypes of ABA-deficient mutants have implicated an essential function of endogenous ABA in the promotion of leaves, stems, cotyledons, primary roots, and lateral roots in the absence of exogenous stress conditions (Koornneef et al., 1982
; Nambara et al., 1998
; Zeevaart and Creelman, 1988
; Cheng et al., 2002
). Similarly, characterization of the ABA-insensitive mutant abi3–6 indicates that ABA signaling is required to promote lateral root formation in response to auxin signals (Brady et al., 2003
). However, raising the ABA level of wild-type plants with exogenously supplied ABA has the opposite effect: lateral root formation is inhibited (De Smet et al., 2003
). Thus, ABA signaling can play both a positive and a negative role in Arabidopsis lateral root formation.
Metabolic or nutritional signals, such as nitrate levels (Signora et al., 2001
) or the nitrogen: carbon ratio (N : C ratio) (Malamy and Ryan, 2001
) also regulate lateral root formation. ABA is required to mediate the regulatory effects of nitrate on root branching, signaling through a portion of the ABA response pathway comprising ABI4 and ABI5. Other ABA signaling genes that play a strong role in seed germination, such as ABI1, ABI2 and ABI3, are not required for the lateral root response, indicating a branched pathway (Signora et al., 2001
). Interestingly, although auxin has been thought to be the primary regulator of lateral root formation, the ABA-induced inhibition of lateral root development appears to be auxin-independent (De Smet et al., 2003
). Thus, one function of ABA in regulating root architecture is to mediate nutritional signals in an auxin-independent manner.
Legumes are one of the largest and most widespread angiosperm families, perhaps due to their unique ability to form a symbiosis with nitrogen-fixing soil bacteria, collectively known as Rhizobium. Many of the more than 18 000 legume species form a symbiosis with Rhizobium, but only one nonlegume does, Parasponia (Pueppke and Broughton, 1999
). Doyle and colleagues have proposed that nodulation evolved independently three times within the legume lineage, suggesting the existence of a trait evolved by the common ancestor of this lineage that could predispose legumes to accommodate its rhizobial partner (Doyle et al., 1997
; Doyle, 1998
). A broader analysis including nonlegumes that enter into a nitrogen-fixing symbiosis with the actinomycete, Frankia, proposed the existence of a larger, nitrogen-fixing clade within the rosid I subclass, suggesting an earlier single origin for a predisposition for nitrogen-fixing symbioses in roots (Soltis et al., 1995
). The physiological or developmental characteristics of legumes and actinorhizal plants that lead to a predisposition for nitrogen-fixing root nodules are currently unknown.
In this report, we compare the effect of ABA on lateral root density (LRD), defined as the number of lateral roots per unit of primary root length, in legumes and nonlegumes. We find that, in general, nonlegumes like Arabidopsis respond to ABA by decreasing LRD and legumes respond to ABA by increasing LRD. We examined nodulating and non-nodulating legumes associated with the different predicted origins of nodulation and found that they all responded by increasing LRD in response to ABA, with the exception of Chamaecrista fasciculata (Michx.) Greene and the basal legume, Cercis occidentalis Torr. & A. Gray, which neither increase nor decrease LRD. We also show that increasing LRD in response to ABA is associated with the formation of nonlegume root nodules in the actinorhizal plant Casuarina glauca Sieber ex Spreng., but not in the related, non-nodulating Betula papyrifera Marshall. Our data provide the first evidence for a physiological trait associated with root nodule formation in both legumes and an actinorhizal plant. We speculate that increased lateral root formation in response to ABA is a necessary prerequisite or response to nodule formation.
MATERIALS AND METHODS
Plant growth conditions
Plants for lateral-root assays were grown on sterile media in square petri plates, sealed with surgical tape, and were placed at a near vertical position in a Conviron growth chamber (model MTR30; Winnipeg, Manitoba, Canada). Seed sources are detailed in Table 1. Arabidopsis thaliana and Lotus japonicus were grown on 10 x 10 cm petri dishes. Other species were grown on 25 x 25 cm petri dishes.
|
), lacking nitrogen and sugar, or Murashige and Skoog (MS)/sucrose medium (pH 5.8), containing 1x MS salts (Murashige and Skoog, 1962
), 0.5% (w/v) sucrose, and 1x Gamborg's vitamins (Sigma, St. Louis, Missouri, USA). For ABA treatment, ABA (A4906; Sigma, St. Louis, Missouri, USA) was added to the medium at the specified concentration (0.01, 0.1, 1, 10 µM) before autoclaving. Growth chambers were kept at 20°C, 16-h days, with a light intensity of 195 µE m–2·s–1 and 50% humidity.
Seed germination of nonlegumes
Arabidopsis thaliana, Lactuca sativa, Anethum graveolens, Lycopersicon esculentum, Eschscholzia californica, Casuarina glauca, and Betula papyrifera seeds were surface sterilized in 30% bleach (equivalent to 2% sodium hypochlorite) for 15–20 min, then washed thoroughly with sterile water. Arabidopsis thaliana, L. sativa, A. graveolens, L. esculentum, and E. californica seeds were evenly distributed on plates containing MS/suc or BNM medium. Arabidopsis thaliana seeds were placed at 4°C in the dark for 2 days, then moved to continuous cool light at an intensity of 100 µE m–2·s–1 at room temperature to promote synchronous germination. Lactuca sativa, A. graveolens, L. esculentum, and E. californica seeds were germinated at room temperature in the dark for 2 days. After germination, seeds were transferred to new plates containing the same medium and moved to the growth chamber. Casuarina glauca and Betula papyrifera seeds were soaked in water overnight, then placed at 4°C for at least 3 days. They were subsequently germinated in a damp, inverted petri dish sealed with parafilm under continuous cool light at room temperature.
Seed germination of legumes
Scarification
Most legume seeds require scarification prior to surface sterilization. Mimosa pudica was soaked in hot water (60°C) for 20 min. Other species were scarified in concentrated sulfuric acid for varying lengths of time: Chamaecrista fasciculata for 30 s, Senna hebecarpa for 5 min, Medicago truncatula for 15 min, Lotus japonicus and Lupinus sericeus for 20 min, and Cladrastis lutea and Cercis occidentalis for 60 min.
Surface sterilization
After scarification, seeds were rinsed thoroughly with sterile water and surface sterilized as for Arabidopsis. Commercial Medicago sativa seeds did not require scarification and were placed directly in 70% ethanol for 30–45 min followed by 30–45 min in full-strength bleach. Lotus japonicus seeds were surface sterilized in 70% ethanol, 3% hydrogen peroxide for 10 min.
Germination
Following surface sterilization, seeds were imbibed in sterile water for 3 h on a shaker at room temperature. Seeds were germinated in the dark at room temperature in a damp, inverted petri dish sealed with parafilm. Seedlings were transferred to 25 x 25 cm petri dishes and moved to the growth chamber.
Phylogenetic analysis
We took two approaches to assess the phylogenetic relationships of the sampled taxa. First, we assembled rbcL sequences for our sample taxa when possible or alternatively for their nearest relatives for which rbcL sequences are available (Appendix). Due to sequence length variability in legume rbcL genes, the sequences were trimmed; we used a 1364-bp sequence common to all taxa. These sequences were subjected to phylogenetic analysis using PAUP*, version 3.1 (Swofford, 1993
). Eschscholzia californica was used as an outgroup to root the phylogenetic tree given its basal position in multiple gene phylogenies (Soltis et al., 1995
). We conducted a maximum-parsimony analysis using a heuristic search with tree bisection reconnection (TBR), using 100 replications, holding one tree at each step.
Second, we looked at the placement of our sampled taxa in the three-sequence angiosperm phylogeny of Soltis et al. (1995)
and the rbcL legume phylogeny of Doyle (Doyle et al., 1997
; Savolainen et al., 2000
).
Data analysis
Primary root growth after germination was assessed by marking the position of the root tip at the time of plating, then measuring the distance from that mark to the root tip 20 days later. At that time, lateral roots longer than 0.5 cm were counted. Senna hebecarpa plants were too large to be grown in our culture conditions for such a long period, and thus root length and root number for that species were assessed at 10 days after plating. These measurements were repeated for at least 24 plants from a total of two independent experiments, with the exception of Cladrastis lutea, for which a total of only 10 plants were measured from two different experiments.
Student's t test was performed to test whether two treatment groups differed significantly from each other (Figs. 2 and 3). Fisher's least significant difference test was conducted to perform multiple comparisons for determining which mean scores were significantly different (Figs. 1 and 5).
|
|
|
|
The following results are organized as a report of the experiments with the logic underlying the experiments. Within the scope of this study, we wanted to reveal any trend in the response of root architecture to ABA in legumes and nonlegumes rather than to fully characterize the root response of legumes to ABA.
ABA stimulates lateral root formation in Medicago truncatula
Arabidopsis thaliana seedlings grown on ABA concentrations ranging from 0.1 to 1 µM produced fewer lateral roots (De Smet et al., 2003
). In contrast, we observed that Medicago truncatula seedlings treated with ABA formed more lateral roots. As we increased the concentration of ABA, the average number of lateral roots increased by 58% at 1 µM and 75% at 10 µM ABA (Fig. 1B). At these concentrations, the primary root length decreased by 22% at 1 µM and 38% at 10 µM ABA (Fig. 1A). Thus, the LRD (number of lateral roots/cm primary root length) increased by one-fold at 1 µM and two-fold at 10 µM ABA (Fig. 1C). Because 1 µM ABA is the lowest concentration at which a significant change in LRD occurs in M. truncatula, we selected 1 µM as the standard ABA concentration in all subsequent experiments, so that the effect of ABA on root architecture could be examined while minimally affecting other aspects of plant physiology.
In our experiments, M. truncatula was grown on BNM medium, which contains no sugar or nitrogen. However, in the study by De Smet and colleagues (2003)
, A. thaliana seedlings were grown on MS/suc medium. BNM and MS/suc media differ not only in carbon content (0 in BNM and 0.5% sucrose in supplemented MS medium) but also in fixed nitrogen: BNM medium lacks nitrogen, whereas MS/suc medium contains 92 mM NH4+ and 57 mM nitrate (De Smet et al., 2003
). As the carbon : nitrogen (C : N) ratio in the medium has been reported to affect lateral root formation (Coruzzi and Bush, 2001
; Coruzzi and Zhou, 2001
; Malamy and Ryan, 2001
), differences in the composition of the media could underlie the differences between our observations of M. truncatula root architecture and the published report of ABA on A. thaliana. To test whether the growth medium used accounts for differences in root responsiveness to ABA, we grew both A. thaliana and M. truncatula plants on MS/suc or BNM media and measured primary root length and lateral root number at 9 days in order to compare our results with the De Smet study. Arabidopsis seedlings grown on MS/suc responded to ABA by decreasing LRD by 36% (Fig. 2A), confirming the observation of De Smet and colleagues (De Smet et al., 2003
). We found that A. thaliana seedlings grown on BNM medium responded even more strongly to ABA, decreasing LRD by 62% (Fig. 2A). In contrast, M. truncatula seedlings grown on either MS/suc or BNM medium responded to ABA by increasing lateral root number approximately two- or six-fold, respectively (Fig. 2B). Our results indicate that the composition of the media is not the reason for the opposing effects of ABA on Arabidopsis and M. truncatula root architecture, but rather that Arabidopsis and M. truncatula roots differ in their physiological response to ABA.
ABA has opposite effects on the root architecture of legumes and nonlegumes independent of the ability to nodulate
To determine whether other species respond to ABA like M. truncatula or A. thaliana, we examined the effect of ABA on LRD for a selected set of legumes and nonlegumes. Arabidopsis thaliana, Anethum graveolens, Lactuca sativa cv. Iceberg, Lycopersicon esculentum cv. Sweetie, and Eschscholzia californica were chosen as representative nonlegume species, and M. truncatula, Lotus japonicus, M. sativa, and Lupinus sericeus as representative legume species. We found that the nonlegumes, A. graveolens, Lactuca sativa, Lycopersicon esculentum, and E. californica all respond to ABA by decreasing LRD just as Arabidopsis does, whereas Lotus japonicus, Lupinus sericeus, and M. sativa respond to ABA by increasing LRD like M. truncatula (Fig. 3). Interestingly, we found that although 1 µM ABA decreased the primary root length of most legume species, it had no significant effect on the primary root length of the nonlegume species tested (data not shown), indicating that the change in LRD for nonlegumes is due entirely to the change in lateral root number. Because this concentration of ABA had no measurable effect on nonlegume primary root growth and the shoots appeared vigorous and green, the health of these plants does not appear to have been compromised by the ABA treatment. Thus, the altered LRD in legumes and nonlegumes appears to be an altered response of root architecture to ABA, with ABA exerting an opposite effect on lateral root development in legumes and nonlegumes.
Nodulation is predicted to have evolved at least three times within the legume family (Doyle et al., 1997
; Doyle, 1998
); the species examined in our initial analysis, M. truncatula, M. sativa, Lotus japonicus, and Lupinus sericeus, all stem from the first predicted origin of nodulation (Fig. 4). To test whether nodulating legumes from the other two predicted origins have the same response as M. truncatula, we examined the effect of ABA on species from the second and third origins of nodulation, Mimosa pudica and Chamaecrista fasciculata, respectively (Fig. 4). We observed that M. pudica responded to ABA by increasing LRD like other legumes (Fig. 3). In contrast, C. fasciculata did not significantly change LRD in response to 1 µM ABA. Upon closer analysis, we found that the response of C. fasciculata to ABA is dependent on seed quality, with tan seeds responding to ABA by increasing lateral root number and black seeds responding by decreasing lateral root number. This response of lateral root formation to ABA also correlated with ease of germination. Tan seeds had generally poor germination, while black seeds germinated easily (data not shown). In summary, our results suggest that an altered ABA response correlates with nodule formation in legume species arising from two of the putative origins of nodulation. ABA responsiveness of C. fasciculata lateral roots is less clear and may be influenced by seed history or seed maturity, an ABA-dependent process.
|
Root architecture in an actinorhizal plant has a legume-like response to ABA
In addition to the Rhizobium-legume symbiosis, the other major nitrogen-fixing symbiosis involving higher plants is between the actinomycete Frankia and several species of mostly woody dicotyledonous plants that form four well-supported clades in the rosid I clade (Swensen, 1996
). To test whether the effect of ABA on LRD in actinorhizal plants resembles that of legumes or nonlegumes, we examined lateral root formation in the actinorhizal species Casuarina glauca. We found that C. glauca responds to ABA by increasing LRD just as legumes do (Fig. 3). However, the related nonactinorhizal species, Betula papyrifera, responds to ABA by decreasing LRD in a manner consistent with other nonlegumes (Fig. 3). These results indicate that altered ABA responsiveness of LRD in C. glauca correlates with nodule formation in this actinorhizal lineage.
Response of lateral root development to ABA in legumes and nonlegumes differs over a wide range of concentrations
Our results indicate that most legumes respond to 1 µM ABA by increasing LRD, whereas nonlegumes respond to the same concentration of ABA by decreasing LRD. We hypothesized that the opposite responses of legumes and nonlegumes to ABA might be due to a differential responsiveness of lateral root formation to ABA in these species. To test this, we measured the LRD of two additional legume species (Medicago sativa and Mimosa pudica) and three representative nonlegumes (Arabidopsis thaliana, Lactuca sativa, and Anethum graveolens) in response to different concentrations of ABA (Fig. 5). We found that M. sativa and M. pudica respond to increasing concentrations of ABA by increasing LRD as M. truncatula does (Figs. 1, 5A, B). In contrast, LRD in the nonlegumes A. thaliana, A. graveolens, and L. sativa were not stimulated by ABA even at low concentrations (0.01 and 0.1 µM), while higher concentrations of ABA reliably inhibited LRD in all three species (Fig. 5). The apparent stimulation of LRD in A. thaliana and A. graveolens is not statistically significant. Thus, our data indicate that exogenously applied ABA has opposite effects on lateral root formation in legumes and nonlegumes over a wide range of concentrations.
Phylogenetic analysis
To compare the phylogenetic relationships of the sampled taxa, a phylogeny was constructed based on rbcL sequences (Fig. 4). Our own analysis of deeper phylogenetic relationships was congruent with the Savolainen et al. (2000)
three-sequence phylogeny. Our analysis of the more recent phylogenetic events involving the legumes in our study set is entirely congruent with the phylogeny for the legumes based on rbcL for the Leguminosae (Doyle et al., 1997
). We found that the physiological responses we observed in our study set are congruent with the phylogenetic relationships of the sampled taxa. That is, all nodulating and non-nodulating legume taxa associated with three origins of nodulation increased LRD in response to ABA with the exception of Chamaecrista fasciculata. Cercis occidentalis, a basal legume that does not nodulate, has an intermediate response partway between that of nonlegumes and legumes. Finally, the actinorhizal nodulator, Casuarina glauca, but not its non-actinorhizal sister taxa, Betula papyrifera, also responds to ABA by increasing LRD, just as legume taxa associated with nodulation do.
DISCUSSION
Since the identification of ABA in the early 1960s, much effort has been devoted to understanding its function (Rock, 2000
), but its effect on lateral root development has only recently been observed. ABA was thought to act as an inhibitor for lateral root development, because application of exogenous ABA (0.1–1 µM) inhibits lateral root number in Arabidopsis (De Smet et al., 2003
). However, genetic analysis indicate a growth-promoting function of ABA on lateral roots (Cheng et al., 2002
). Our results represent the first direct evidence that exogenous ABA promotes lateral root development. In this paper we show that 1 µM ABA increases LRD in most legumes, which suggests that ABA may be able to serve as a growth-promoting hormone for lateral root development in legumes. This stimulation of legume lateral-root development by ABA represents a novel ABA response. Our phylogenetic analysis of this trait is consistent with a single acquisition of altered LRD ABA responsiveness early in the legume lineage and again in a nodulating actinorhizal species.
Responsiveness of root branching to ABA is correlated with nodule formation in legumes
Nodulation is inferred to have evolved at least three times within the legume lineage (Doyle et al., 1997
). The independent evolution of nodulation three times within the Fabaceae suggests that legumes have acquired some physiological or developmental trait that predisposes them to form a symbiosis with Rhizobium. Current models suggest that a predisposition for nodulation originated only once within the legume lineage (Doyle, 1998
). The nature of this predisposition is unknown, but differences in secondary metabolites or signaling molecules (such as flavonoids), altered defense responses, novel receptor molecules or cell wall components, and different phytohormones or phytohormone sensitivities have all been proposed as possible predisposing factors (Doyle et al., 1997
; Doyle, 1998
; Hirsch et al., 2001
).
In this study, we have identified a physiological trait, increased lateral root formation in response to ABA, which marks all nodulating legumes in our study set with the exception of Chamaecrista fasciculata. In addition, we observed that two non-nodulating legume species, Cladrastis lutea and Senna hebecarpa, closely related to nodulating species, exhibit the same responses to ABA as legumes that form a rhizobial symbiosis. Cercis occidentalis, a basal legume that fails to form root nodules, exhibits an intermediate response to ABA, partway between the nonlegumes, which decrease LRD, and other legumes, which increase LRD. Together, these data suggest that altered LRD responsiveness to ABA is widespread in the Fabaceae, but not in the rest of the angiosperms, and are consistent with a single acquisition of this physiological trait early in the legume lineage, followed by a single loss in the branch leading to Chamaecrista (Fig. 5).
The most parsimonious interpretation of our data is that the acquisition of the ABA trait occurred early in the legume lineage, prior to the three predicted origins of nodulation. The acquisition of this ABA trait is coincident with the appearance of a predisposition for nodulation within the legumes (Fig. 5) (Doyle, 1998
). Altered phytohormone production or sensitivity is one of many physiological traits that has been suggested as a possible predisposing event for nodule formation (Doyle et al., 1997
; Doyle, 1998
; Hirsch et al., 2001
). Thus, the altered responsiveness of lateral root formation to ABA might have been one of the events that predisposed nodulating legumes to form nitrogen-fixing nodules. Alternatively, altering the responsiveness of lateral root formation to ABA might have been a compensatory change in response to the appearance of nodules. If this is the case, then our data suggest a single origin for the nodulation of legumes, with subsequent losses in other lineages. This model is only slightly less parsimonious than the predicted three-origin model. If losses of nodulation are easier to achieve than gains, then a single origin for nodulation would be more likely (Doyle, 1998
). In fact, such a model is consistent with the observation that mutations in single genes confer a complete Nod– phenotype (Peterson and Barnes, 1981
; Sagan et al., 1995
; Schauser et al., 1998
; Szczyglowski et al., 1998
; Catoira et al., 2000
).
The loss of the ABA trait in the nodulating Chamaecrista fasciculata indicates that this trait is not absolutely required for nodulation. Therefore we propose a model in which acquisition of the ABA trait was a necessary step in the establishment of nodulation but was no longer needed once nodulation had evolved, much as a scaffold is necessary during the construction of a building, but is no longer required once the building is erected. By this model, loss of the ABA trait in Chamaecrista could not result in the loss of nodulation, since the ability to nodulate was already established in this species.
The link between lateral root formation and nodulation in legumes
The hypothesis that altered phytohormone sensitivity of developing lateral roots could affect legume nodulation implies a connection between legume lateral root development and nodule formation. Similarities in nodule and lateral root development suggest that they may share a common evolutionary origin (Hirsch and LaRue, 1997
). Actinorhizal nodules and nodules formed on the roots of Parasponia are considered to be modified lateral roots. Legume nodules, however, differ from lateral roots in origin, level of DNA endoreduplication, and final structure, but they share many features (Hirsch, 1992
), especially positioning at sites normally occupied by lateral roots, e.g., opposite protoxylem points of the primary vascular system (Hirsch and LaRue, 1997
), or initiating in the mature root at sites of lateral root emergence (Mathesius et al., 2000
). In addition, aborted nodules have been observed to revert to lateral roots (Ferraioli et al., 2004
). Finally, recent analysis of the latd/nip mutants provides the first genetic evidence for shared components between lateral root and nodule development (Veereshlingam et al., 2004
; Bright et al., 2005
).
A physiological balance between lateral roots and nodules was first proposed by Nutman (1948)
, based on his observation that nodulated clover plants had fewer lateral roots than uninoculated plants. More recently the supernodulating mutant, har1, of Lotus japonicus, has been shown to form excess nodules when inoculated with Rhizobium, but excess lateral roots when left uninoculated (Wopereis et al., 2000
). Subsequent research identified plant hormones that have opposite effects on nodule and lateral root formation. Decreasing auxin levels in alfalfa roots by adding auxin transport inhibitors can lead to the formation of nodule-like structures (Allen et al., 1953
; Hirsch et al., 1989
), whereas increasing auxin levels stimulates lateral root formation (Casimiro et al., 2003
). In contrast, locally applied cytokinin triggers nodule formation, but inhibits lateral root formation (Cooper and Long, 1994
; Lohar et al., 2004
). The gaseous hormone ethylene clearly inhibits legume nodulation (Guinel and Geil, 2002
), but has not been reported to affect lateral root formation, although it has been shown to stimulate adventitious root formation (Clark et al., 1999
; Mergemann and Sauter, 2000
). Similarly, the nutrient nitrate has been shown to locally stimulate lateral root formation but systemically inhibit nodulation (Carroll and Gresshoff, 1983
; Zhang and Forde, 1998
). Thus, the opposed regulation of nodules and lateral roots appears to be a common theme.
In this study, we demonstrated that ABA increases LRD in nodulating legumes. Coincidentally, exogenous ABA inhibits root nodule formation after inoculation in Pisum sativum (Phillips, 1971
), Glycine max (Bano et al., 2002
), Trifolium repens, and Lotus japonicus (Suzuki et al., 2004
) and in Medicago truncatula (Liang and Harris, unpublished data). Moreover, endogenous ABA levels have been shown to be high in nodules of P. sativum (Charbonneau and Newcomb, 1985
), G. max (Williams and Sicardi de mallorca, 1982
), and the actinorhizal plant Alnus glutinosa (Watts et al., 1983
). Thus, ABA may represent one of the mechanisms by which the balance between lateral roots and nodules on a legume root is maintained.
Responsiveness of root branching to ABA is correlated with nodule formation in the actinorhizal plant, Casuarina glauca
Actinorhizal plants are nonlegumes that form symbiotic root nodules with the gram-positive microbe, Frankia. Phylogenetic analysis demonstrates that the flowering plant families involved in rhizobial or actinorhizal symbioses belong to a single nitrogen-fixing clade in the rosid I lineage, implying a single origin for the predisposition for symbiotic nitrogen-fixation in the common ancestor of legumes and actinorhizal plants (Soltis et al., 1995
). Although both actinorhizal and legume nodules share a similar function, housing nitrogen-fixing bacterial symbionts, the origin and structure of these two nodule types are quite distinct. Comparison of these two root symbioses may lead to a greater understanding of the plant requirements for the accommodation of nitrogen-fixing symbionts (Vessey et al., 2004
). Actinorhizal nodules are clearly modified lateral roots with a central vasculature and a meristem that can, in some species, revert to a lateral root meristem (Hirsch and LaRue, 1997
). Based on significant morphological differences between actinorhizal nodules from different clades in conjunction with molecular evidence, actinorhizal symbioses have been suggested to have evolved between four and six times within the rosid I lineage (Swensen, 1996
). We found that Casuarina glauca, an actinorhizal plant from clade IV, has the same physiological trait as legumes, namely increased LRD in response to ABA. The LRD of the closely related non-actinorhizal species, Betula papyrifera, however, has an ABA sensitivity that resembles other nonlegumes. It is intriguing that roots that form an actinorhizal symbiosis, which clearly has an origin independent from the Rhizobium-legume symbiosis, still have the altered LRD regulation by ABA that characterizes nodulating legume roots. The presence of this trait in a nodulating, actinorhizal species provides strong evidence to correlate altered lateral root regulation by ABA with the appearance of symbiotic root nodules. The absence of the LRD/ ABA trait in the non-actinorhizal B. papyrifera may indicate that this trait was acquired after the lineages including C. glauca and B. papyrifera diverged. Alternatively, this altered response to ABA might have been acquired coincidently with a predisposition for nodulation in a common ancestor to both the Fabaceae and the four actinorhizal clades and then lost in the B. papyrifera lineage. However, to adequately test this model, we have to examine additional actinorhizal species.
Our identification of a physiological trait coincident with the acquisition of a predisposition for symbiotic nodule formation provides the first glimpse of the physiological and developmental changes that had to occur in plant roots to accommodate a bacterial endosymbiont. Although it is not clear whether the altered ABA root response was acquired before or after the ability to nodulate, the extensive correlation between nodulating taxa and an altered ABA response suggests that this physiological change in host roots plays an important role in nodulation both in legumes and in at least one nonlegume. Further analysis of the effect of ABA and altered root architecture on the ability of plants to nodulate should provide more insight into the physiological changes that accompanied the evolution of symbiotic nitrogen-fixation in flowering plants.
.
. Bousquet, J., S. H. Strauss, and P. Li. 1992. Complete congruence between morphological and rbcL-based molecular phylogenies in birches and related species (Betulaceae). Molecular Biology and Evolution 9: 1076–1088.
Doyle, J. J., J. L. Doyle, J. A. Ballenger, E. E. Dickson, T. Kajita, and H. Ohashi. 1997. A phylogeny of the chloroplast gene rbcL in the Leguminosae: taxonomic correlations and insights into the evolution of nodulation. American Journal of Botany 84: 541–554.
Hoot, S. B., J. W. Kadereit, F. R. Blattner, K. B. Jork, A. E. Schwarzbach, and P. R. Crane. 1997. Data congruency and phylogeny of the papaveraceae s.l. based on four data sets: atpB and rbcL sequences, trnK restriction sites, and morphological characters. Systematic Botany 22: 575– 590.
Kaess, E., and M. Wink. 1995. Molecular phylogeny of the Papilionoideae (Family Leguminosae): rbcL sequences versus chemical taxonomy. Acta Botanica Croatica 108: 149–162.
Kato, T., T. Kaneko, S. Sato, Y. Nakamura, and S. Tabata. 2000. Complete structure of the chloroplast genome of a legume, Lotus japonicus. DNA Research 7: 323–330.
Olmstead, R. G., H. J. Michaels, K. M. Scott, and J. D. Palmer. 1992. Monophyly of the Asteridae and identification of their major lineages inferred from DNA sequences of rbcL. Annals of the Missouri Botanical Garden 79: 249–265.
Olmstead, R. G., B. Bremer, K. M. Scott, and J. D. Palmer. 1993. A parsimony analysis of the Asteridae sensu lato based on rbcL sequences. Annals of the Missouri Botanical Garden 80: 700–722.
Sato, S., Y. Nakamura, T. Kaneko, E. Asamizu, and S. Tabata. 1999. Complete structure of the chloroplast genome of Arabidopsis thaliana. DNA Research 6: 283–290.
Sogo, A., H. Setoguchi, J. Noguchi, T. Jaffre, and H. Tobe. 2001. Molecular phylogeny of Casuarinaceae based on rbcL and matK gene sequences. Journal of Plant Research 114: 459–464.
FOOTNOTES
1 The authors thank Dave Barrington for advice, encouragement, and suggestions on the phylogenetic portion of this analysis and for comments on the manuscript; S. Long for Medicago sativa seeds; R. Calloway for Lupinus sericeus seeds; K. Szczglowski and F. de Bruijn for Lotus japonicus seeds; M. Tierney for Arabidopsis thaliana (Columbia) seeds; H. Driscoll for advice on using PAUP*; members of our laboratory for helpful discussions on this analysis; and the anonymous reviewers for their comments and suggestions. This work was supported by National Science Foundation grant IBN-0212992 and by U.S. Department of Agriculture Experiment Station grant no. VT-BO-00804 to J.M.H. 
2 Author for correspondence (e-mail: Jeanne.Harris{at}uvm.edu
)
LITERATURE CITED
Allen E. K. O. N. Allen A. S. Newman 1953 Pseudonodulation of leguminous plants induced by 2-bromo-3,5-dichlorobenzoic acid. American Journal of Botany 40: 429-435[CrossRef][Web of Science]
Bano A. J. E. Harper R. M. Auge D. S. Neuman 2002 Changes in phytohormone levels following inoculation of two soybean lines differing in nodulation. Functional Plant Biology 29: 965-974[Web of Science]
Brady S. M. S. F. Sarkar D. Bonetta P. McCourt 2003 The ABSCISIC ACID INSENSITIVE 3 (ABI3) gene is modulated by farnesylation and is involved in auxin signaling and lateral root development in Arabidopsis. Plant Journal 34: 67-75[CrossRef][Web of Science][Medline]
Bright L. J. Y. Liang D. M. Mitchell J. M. Harris 2005 The LATD gene of Medicago truncatula is required both for nodule and root development. Molecular Plant–Microbe Interactions 18: 521-532[CrossRef]
Carroll B. J. P. M. Gresshoff 1983 Nitrate inhibition of nodulation and nitrogen fixation in white clover. Zeitschrift fur Botanik 110: 77-88
Casimiro I. T. Beeckman N. Graham R. Bhalerao H. Zhang P. Casero G. Sandberg M. J. Bennett 2003 Dissecting Arabidopsis lateral root development. Trends in Plant Science 8: 165-171[CrossRef][Web of Science][Medline]
Catoira R. C. Galera F. de Billy R. V. Penmetsa E.-P. Journet F. Maillet C. Rosenberg D. Cook C. Gough J. Dénarié 2000 Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway. Plant Cell 12: 1647-1666
Charbonneau G. A. W. Newcomb 1985 Growth regulators in developing effective root nodules of the garden pea (Pisum sativum L). Biochemie und Physiologie der Pflanzen 180: 667-681[Web of Science]
Cheng W. H. A. Endo L. Zhou J. Penney H. C. Chen A. Arroyo P. Leon E. Nambara T. Asami M. Seo T. Koshiba J. Sheen 2002 A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14: 2723-2743
Clark D. G. E. K. Gubrium J. E. Barrett T. A. Nell H. J. Klee 1999 Root formation in ethylene-insensitive plants. Plant Physiology 121: 53-60
Cooper J. B. S. R. Long 1994 Morphogenetic rescue of Rhizobium meliloti nodulation mutants by trans-zeatin secretion. Plant Cell 6: 215-225[Abstract]
Coruzzi G. D. R. Bush 2001 Nitrogen and carbon nutrient and metabolite signaling in plants. Plant Physiology 125: 61-64
Coruzzi G. M. L. Zhou 2001 Carbon and nitrogen sensing and signaling in plants: emerging ‘matrix effects’. Current Opinion in Plant Biology 4: 247-253[CrossRef][Web of Science][Medline]
De Smet I. L. Signora T. Beeckman D. Inzé C. H. Foyer H. Zhang 2003 An abscisic acid-sensitive checkpoint in lateral root development of Arabidopsis. Plant Journal 33: 543-555[CrossRef][Web of Science][Medline]
Doyle J. J. 1998 Phylogenetic perspectives on nodulation: evolving views of plants and symbiotic bacteria. Trends in Plant Science 3: 473-478[CrossRef][Web of Science]
Doyle J. J. J. L. Doyle J. A. Ballenger E. E. Dickson T. Kajita H. Ohashi 1997 A phylogeny of the chloroplast gene rbcL in the Leguminosae: taxonomic correlations and insights into the evolution of nodulation. American Journal of Botany 84: 541-554[Abstract]
Ehrhardt D. W. E. M. Atkinson S. R. Long 1992 Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors. Science 256: 998-1000
Ferraioli S. R. Tatè A. Rogato M. Chiurazzi E. J. Patriarca 2004 Development of ectopic roots from abortive nodule primordia. Molecular Plant–Microbe Interactions 17: 1043-50[CrossRef]
Finkelstein R. R. S. S. Gampala C. D. Rock 2002 Abscisic acid signaling in seeds and seedlings. Plant Cell 14: (Supplement) S15-45
Giraudat J. F. Parcy N. Bertauche F. Gosti J. Leung P. C. Morris M. Bouvier-Durand N. Vartanian 1994 Current advances in abscisic acid action and signalling. Plant Molecular Biology 26: 1557-1577[CrossRef][Web of Science][Medline]
Guinel F. C. R. D. Geil 2002 A model for the development of the rhizobial and arbuscular mycorrhizal symbioses in legumes and its use to understand the roles of ethylene in the establishment of these two symbioses. Canadian Journal of Botany 80: 695-720[CrossRef]
Himmelbach A. M. Iten E. Grill 1998 Signalling of abscisic acid to regulate plant growth. Philosophical Transactions of the Royal Society of London, B, Biological Sciences 353: 1439-1444[CrossRef]
Hirsch A. 1992 Tansley review no. 40. Developmental biology of legume nodulation. New Phytologist 122: 211-237[CrossRef][Web of Science]
Hirsch A. T. Bhuvaneswari J. Torrey T. Bisseling 1989 Early nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport inhibitors. Proceedings of the National Acadamy of Sciences, USA 86: 1244-1248
Hirsch A. M. T. A. LaRue 1997 Is the legume nodule a modified root or stem or an organ sui generis?. Critical Reviews in Plant Sciences 16: 361-392[CrossRef][Web of Science]
Hirsch A. M. M. R. Lum J. A. Downie 2001 What makes the rhizobia-legume symbiosis so special?. Plant Physiology 127: 1484-1492
Koornneef M. M. L. Jorna D. L. C. Brinkhorst-van der Swan C. M. Karssen 1982 The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana (L.) Heynh. Theoretical and Applied Genetics 61: 385-393[Web of Science]
Lohar D. P. J. E. Schaff J. G. Laskey J. J. Kieber K. D. Bilyeu D. M. Bird 2004 Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses. Plant Journal 38: 203-214[CrossRef][Web of Science][Medline]
López-Bucio J. A. Cruz-Ramírez L. Herrera-Estrella 2003 The role of nutrient availability in regulating root architecture. Current Opinion in Plant Biology 6: 280-287[CrossRef][Web of Science][Medline]
Malamy J. E. 2005 Intrinsic and environmental response pathways that regulate root system architecture. Plant, Cell & Environment 28: 67-77[CrossRef][Medline]
Malamy J. E. K. S. Ryan 2001 Environmental regulation of lateral root initiation in Arabidopsis. Plant Physiology 127: 899-909
Mathesius U. J. J. Weinman B. G. Rolfe M. A. Djordjevic 2000 Rhizobia can induce nodules in white clover by "hijacking" mature cortical cells activated during lateral root development. Molecular Plant– Microbe Interactions 13: 170-182
Mergemann H. M. Sauter 2000 Ethylene induces epidermal cell death at the site of adventitious root emergence in rice. Plant Physiology 124: 609-614
Murashige T. F. Skoog 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15: 473-497[CrossRef]
Nambara E. H. Kawaide Y. Kamiya S. Naito 1998 Characterization of an Arabidopsis thaliana mutant that has a defect in ABA accumulation: ABA-dependent and ABA-independent accumulation of free amino acids during dehydration. Plant and Cell Physiology 39: 853-858
Nutman P. S. 1948 Physiological studies on nodule formation. I. The relation between nodulation and lateral root formation in red clover. Annals of Botany XII 81-96
Peterson M. A. D. K. Barnes 1981 Inheritance of ineffective nodulation and non-nodulation traits in alfalfa. Crop Science 21: 611-616
Phillips D. A. 1971 Abscisic acid inhibition of root nodule initiation in Pisum sativum. Planta 100: 181-190[CrossRef][Web of Science]
Pueppke S. G. W. J. Broughton 1999 Rhizobium sp. strain NGR234 and R. fredii USDA257 share exceptionally broad, nested host ranges. Molecular Plant–Microbe Interactions 12: 293-318
Rock C. D. 2000 Tansley review no. 120. Pathways to abscisic acid-regulated gene expression. New Phytologist 148: 357-396[CrossRef][Web of Science]
Sagan M. D. Morandi E. Tarenghi G. Duc 1995 Selection of nodulation and mycorrhizal mutants in the model plant Medicago truncatula (Gaertn.) after gamma-ray mutagenesis. Plant Science 111: 63-71[CrossRef][Web of Science]
Savolainen V. M. W. Chase S. B. Hoot C. M. Morton D. E. Soltis C. Bayer M. F. Fay A. Y. de Bruijn S. Sullivan Y. L. Qiu 2000 Phylogenetics of flowering plants based on combined analysis of plastid atpB and rbcL gene sequences. Systematic Biology 49: 306-362[CrossRef][Web of Science][Medline]
Schauser L. K. Handberg N. Sandal J. Stiller T. Thykjaer E. Pajuelo A. Nielsen J. Stougaard 1998 Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus. Molecular Genetics and Genomics 259: 414-423
Sharp R. E. M. E. LeNoble 2002 ABA, ethylene and the control of shoot and root growth under water stress. Journal of Experimental Botany 53: 33-37
Signora L. I. De Smet C. H. Foyer H. Zhang 2001 ABA plays a central role in mediating the regulatory effects of nitrate on root branching in Arabidopsis. Plant Journal 28: 655-662[CrossRef][Web of Science][Medline]
Soltis D. E. P. S. Soltis D. R. Morgan S. M. Swensen B. C. Mullin J. M. Dowd P. G. Martin 1995 Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms. Proceedings of the National Academy of Sciences, USA 92: 2647-2651
Spollen W. G. M. E. LeNoble T. D. Samuels N. Bernstein R. E. Sharp 2000 Abscisic acid accumulation maintains maize primary root elongation at low water potentials by restricting ethylene production. Plant Physiology 122: 967-976
Suzuki A. M. Akune M. Kogiso Y. Imagama K. Osuki T. Uchiumi S. Higashi S. Y. Han S. Yoshida T. Asami M. Abe 2004 Control of nodule number by the phytohormone abscisic acid in the roots of two leguminous species. Plant and Cell Physiology 45: 914-922
Swensen S. M. 1996 The evolution of actinorhizal symbiosis: evidence for multiple origins of the symbiotic association. American Journal of Botany 83: 1503-1512[CrossRef][Web of Science]
Swofford D. L. 1993 PAUP*: phylogenetic analysis using parsimony (*and other methods), version 3.1.1. Smithsonian Institution, Washington, D.C., USA
Szczyglowski K. S. R. Shaw J. Wopereis D. Hamburger S. Copeland F. B. Dazzo F. J. de Bruijn 1998 Nodule organogenesis and symbiotic mutants of the model legume Lotus japonicus. Molecular Plant–Microbe Interactions 11: 684-697[CrossRef]
Veereshlingam H. J. G. Haynes R. V. Penmetsa D. R. Cook D. J. Sherrier R. Dickstein 2004 nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response. Plant Physiology 136: 3692-3702
Vessey J. K. K. Pawlowski B. Bergman 2004 Root-based N2-fixing symbioses: legumes, actinorhizal plants, Parasponia sp. and cycads. Plant and Soil 266: 205-230[CrossRef][Web of Science]
Watts S. H. C. T. Wheeler J. R. Hillman A. M. M. Berrie A. Crozier 1983 Abscisic acid in the nodulated root system of Alnus glutinosa. New Phytologist: 203–208
Williams P. M. Sicardi De Mallorca 1982 Abscisic acid and gibberellin-like substances in roots and root nodules of Glycine max. Plant and Soil 65: 19-26[CrossRef][Web of Science]
Wopereis J. E. Pajuelo F. B. Dazzo Q. Jiang P. M. Gresshoff F. J. De Bruijn J. Stougaard K. Szczyglowski 2000 Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype. Plant Journal 23: 97-114[CrossRef][Web of Science][Medline]
Zeevaart J. A. D. R. A. Creelman 1988 Metabolism and physiology of abscisic acid. Annual Review of Plant Physiology and Plant Molecular Biology 39: 439-473[CrossRef][Web of Science]
Zhang H. B. G. Forde 1998 An Arabidopsis MADS box gene that controls nutrient–induced changes in root architecture. Science 279: 407-409
This article has been cited by other articles:
|
|
 |
|
  Y. Ding, P. Kalo, C. Yendrek, J. Sun, Y. Liang, J. F. Marsh, J. M. Harris, and G. E.D. Oldroyd Abscisic Acid Coordinates Nod Factor and Cytokinin Signaling during the Regulation of Nodulation in Medicago truncatula PLANT CELL, October 1, 2008; 20(10): 2681 - 2695. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
